NEBNext® High-Fidelity 2X PCR

The NEBNext High-Fidelity 2X PCR Master Mix is specifically optimized for the robust, high-fidelity amplification of next-generation sequencing (NGS) libraries, regardless of GC content. The polymerase component of the master mix, Q5™ High-Fidelity DNA Polymerase, is a novel thermostable DNA polymerase that possesses 3´→5´ exonuclease activity, and is fused to a processivity-enhancing Sso7d domain. Q5 High-Fidelity DNA Polymerase also has an ultra-low error rate (> 50-fold lower than that ofTaq DNA Polymerase and 6-fold lower than that of Pyrococcus furiosus (Pfu) DNA Polymerase). The buffer component of the master mix has been optimized for robust amplification, even with GC-rich amplicons.

This combination makes the NEBNext High-Fidelity 2X PCR Master Mix ideal for NGS library construction. This convenient 2X master mix contains dNTPs, Mg⁺⁺ and a proprietary buffer, and requires only the addition of primers and DNA template for robust amplification. When used at the recommended 1X final concentration, the NEBNext High-Fidelity Master Mix contains 2 mM Mg⁺⁺.





Figure 1: Comparative Analysis of Difficult DNA Polymerases with Known Low Coverage Regions.
Indexed libraries were prepared from human IMR90 DNA and split into individual samples for library amplification. Amplification was performed using 8 cycles of PCR with Phusion High-Fidelity DNA Polymerase, KAPA HiFi™ HotStart ReadyMix or NEBNext® High-Fidelity 2X PCR Master Mix. Libraries were sequenced on an Illumina HiSeq® 2000. To correct for slight differences in the number of aligned reads from each library, 180 million reads were randomly extracted from each dataset, representing an average coverage of ~6X. The number of reads overlapping distinct low coverage regions of the human genome (Aird et.al. Genome Biology, 2011) are shown for each library. The NEBNext High-Fidelity 2X PCR Master Mix gives the most optimal performance of the three enzymes / master mixes tested.





Figure 2: Fidelity Comparisons of Different DNA Polymerases.
Fidelity measurements of Taq DNA Polymerase (in Standard Taq Buffer), KAPA HiFi HotStart ReadyMix and NEBNext High-Fidelity 2X PCR Master Mix were measured side-by-side in a PCR-based mutation screening assay using a lacZ method modified from Kermekchiev et al., 2003. Values (n ≥ 2) are expressed relative to KAPA HiFi HotStart ReadyMix. The Q5 High-Fidelity Polymerase in the NEBNext High-Fidelity 2X PCR Master Mix has a level of fidelity >10X higher than that of the polymerase in the KAPA HiFi Hot Start ReadyMix.





Figure 3: Comparative Analysis of Different DNA Polymerases with Genomes of Varying % GC.
Libraries of H. influenza, R. palustris or human genomic DNA were amplified using NEBNext High-Fidelity 2X PCR Master Mix, Phusion High-Fidelity PCR Master Mix with HF Buffer or KAPA HiFi HotStart PCR ReadyMix, and sequenced on an Illumina® HiSeq 2000. GC coverage plots were generated, with % GC content of 100 bp windows on the X axis. Normalized coverage is indicated by the blue circles, windows at GC% indicated by the red lines, and base quality at GC% indicated by the green line. NEBNext High-Fidelity 2X PCR Master Mix provides substantially reduced bias on all three genomic DNA samples.





Figure 4: Comparative Analysis of Different DNA Polymerases at Varying GC %s.
Amplified libraries of human genomic DNA were generated using index primers and NEBNext High-Fidelity 2X PCR Master Mix, Phusion® High-Fidelity PCR Master Mix with HF Buffer or KAPA HiFi HotStart ReadyMix, and sequenced on an Illumina® HiSeq™ 2000. An equal number of reads from each dataset were randomly selected and the percentage of reads was plotted against GC content. NEBNext High-Fidelity 2X PCR Master Mix and KAPA HiF HotStart ReadyMix aligned closely to the expected read frequencies (shaded grey), while Phusion did not.



Source:
An E. coli strain that carries the Q5 High-Fidelity DNA Polymerase gene.

Reaction Conditions:
NEBNext High-Fidelity 2X PCR Master Mix, DNA template and 0.5 μM to 1.25 μM primers (depending on sample input) in a total reaction volume of 50 μl.

Applications:

• Next generation sequencing library construction
• High fidelity PCR
• Difficult amplification
• High-throughput PCR

Enzyme Properties


Heat Inactivation:
No


Reaction & Storage Conditions



Concentration:
2 X

Storage Temperature:
-20°C


Notes


Usage notes:

1. To ensure optimal performance, the master mix should be thawed and resuspended prior to use. Stability testing using up to 20 freeze/thaw cycles has shown no negative effect on master mix performance. The NEBNext High-Fidelity 2X PCR Master Mix may be liquid at -20°C.

FAQs

1. What are the advantages of using NEBNext High-Fidelity 2X PCR Master Mix?
2. Is NEBNext High-Fidelity 2X PCR Master Mix the same as the Q5 High-Fidelity Master Mix?
3. Do I need to make any changes to the cycling conditions for my NGS library amplification I have previously been using with Phusion High-Fidelity PCR Master Mix?
4. How many cycles of PCR should I perform with NEBNext High-Fidelity 2X PCR Master Mix?
5. Will NEBNext High-Fidelity 2X PCR Master Mix incorporate dUTPs?
6. Are the DNA products produced NEBNext High-Fidelity 2X PCR Master Mix blunt-ended or do they have a single base 3’ overhang?

Protocols


Protocols for NEBNext® High-Fidelity 2X PCR Master Mix


Quality Control for Current Lot


Quality control values for a specific lot can be found on the datacard which accompanies each product.

Phosphatase Activity:
Incubation of NEBNext High-Fidelity 2X PCR Master Mix in protein phosphatase assay buffer (1 M diethanolamine @ pH 9.8 and 0.5 mM MgCl₂) containing 2.5 mM p-nitrophenyl phosphate at 37°C for 4 hours yields no detectable p-nitrophenylene anion as determined by spectrophotometric analysis at 405 nm.

Functional Activity (PCR):
30 cycles of PCR amplification of 20 ng genomic DNA in a 50 μl reaction containing 0.5 μM primers and 1X NEBNext High-Fidelity PCR Master Mix result in the expected 737 bp product.

16-Hour Incubation:
A 50 μl reactions containing NEBNext High-Fidelity 2X PCR Master Mix and 1 μg of HindIII digested λ DNA incubated for 16 hours at 37°C results in no detectable non-specific nuclease degradation as determined by agarose gel electrophoresis. 50 μl reactions containing NEBNext High-Fidelity 2X PCR Master Mix and 1 μg of T3 DNA incubated for 16 hours at 37°C results in no detectable non-specific nuclease degradation as determined by agarose gel electrophoresis.


Companion Products


NEBNext® ChIP-Seq Library Prep Reagent Set for Illumina®
NEBNext® DNA Library Prep Master Mix Set for Illumina®
NEBNext® DNA Library Prep Reagent Set for Illumina®
NEBNext® mRNA Library Prep Master Mix Set for Illumina®
NEBNext® mRNA Library Prep Reagent Set for Illumina®
NEBNext® Multiplex Oligos for Illumina (Index Primers Set 1)
NEBNext® Singleplex Oligos for Illumina®


Legal


Licenses/Patents/Disclaimers:
This product is licensed from Bio-Rad Laboratories, Inc. under U.S. Pat. Nos. 6,627,424, 7,541,170, 7,670,808, 7,666,645 and corresponding patents in other countries for use only in: (a) standard (non-real time) PCR in the research field only, but not real-time PCR or digital PCR; (b) any in-vitro diagnostics application, except for applications using real-time or digital PCR; and (c) any non-PCR applications in DNA sequencing, isothermal amplification and the production of synthetic DNA.